Circadian Clock and Hypoxia.

The timing of life on Earth is remarkable: between individuals of the same species, a highly similar temporal pattern is observed, with shared periods of activity and inactivity each day. At the individual level, this means that over the course of a single day, a person alternates between two states. They are either upright, active, and communicative or they lie down in a state of (un)consciousness called sleep where even the characteristic of neuronal signals in the brain shows distinctive properties. The circadian clock governs both of these time stamps-activity and (apparent) inactivity-making them come and go consistently at the same approximate time each day. This behavior thus represents the meeting of two pervasive systems: the circadian clock and metabolism. In this article, we will describe what is known about how the circadian clock anticipates daily changes in oxygen usage, how circadian clock regulation may relate to normal physiology, and to hypoxia and ischemia that can result from pathologies such as myocardial infarction and stroke.

Circadian clocks: It's time for chronobiology.

Circadian clocks are everywhere, yet we still have not translated the vast knowledge gained in the past 20 years on the properties of circadian clocks into practical applications. We are missing opportunities for improving quality of life, health, and sustainability.

The circadian clock of the bacterium B. subtilis evokes properties of complex, multicellular circadian systems.

Circadian clocks are pervasive throughout nature, yet only recently has this adaptive regulatory program been described in nonphotosynthetic bacteria. Here, we describe an inherent complexity in the Bacillus subtilis circadian clock. We find that B. subtilis entrains to blue and red light and that circadian entrainment is separable from masking through fluence titration and frequency demultiplication protocols. We identify circadian rhythmicity in constant light, consistent with the Aschoff's rule, and entrainment aftereffects, both of which are properties described for eukaryotic circadian clocks. We report that circadian rhythms occur in wild isolates of this prokaryote, thus establishing them as a general property of this species, and that its circadian system responds to the environment in a complex fashion that is consistent with multicellular eukaryotic circadian systems.

Rhythms in barriers and fluids: Circadian clock regulation in the aging neurovascular unit.

The neurovascular unit is where two very distinct physiological systems meet: The central nervous system (CNS) and the blood. The permeability of the barriers separating these systems is regulated by time, including both the 24 h circadian clock and the longer processes of aging. An endogenous circadian rhythm regulates the transport of molecules across the blood-brain barrier and the circulation of the cerebrospinal fluid and the glymphatic system. These fluid dynamics change with time of day, and with age, and especially in the context of neurodegeneration. Factors may differ depending on brain region, as can be highlighted by consideration of circadian regulation of the neurovascular niche in white matter. As an example of a potential target for clinical applications, we highlight chaperone-mediated autophagy as one mechanism at the intersection of circadian dysregulation, aging and neurodegenerative disease. In this review we emphasize key areas for future research.

Keeping an eye on circadian time in clinical research and medicine.

BACKGROUND: Daily rhythms are observed in humans and almost all other organisms. Most of these observed rhythms reflect both underlying endogenous circadian rhythms and evoked responses from behaviours such as sleep/wake, eating/fasting, rest/activity, posture changes and exercise. For many research and clinical purposes, it is important to understand the contribution of the endogenous circadian component to these observed rhythms. CONTENT: The goal of this manuscript is to provide guidance on best practices in measuring metrics of endogenous circadian rhythms in humans and promote the inclusion of circadian rhythms assessments in studies of health and disease. Circadian rhythms affect all aspects of physiology. By specifying minimal experimental conditions for studies, we aim to improve the quality, reliability and interpretability of research into circadian and daily (i.e., time-of-day) rhythms and facilitate the interpretation of clinical and translational findings within the context of human circadian rhythms. We describe protocols, variables and analyses commonly used for studying human daily rhythms, including how to assess the relative contributions of the endogenous circadian system and other daily patterns in behaviours or the environment. We conclude with recommendations for protocols, variables, analyses, definitions and examples of circadian terminology. CONCLUSION: Although circadian rhythms and daily effects on health outcomes can be challenging to distinguish in practice, this distinction may be important in many clinical settings. Identifying and targeting the appropriate underlying (patho)physiology is a medical goal. This review provides methods for identifying circadian effects to aid in the interpretation of published work and the inclusion of circadian factors in clinical research and practice.

Deviations from temporal scaling support a stage-specific regulation for C. elegans postembryonic development.

BACKGROUND: After embryonic development, Caenorhabditis elegans progress through for larval stages, each of them finishing with molting. The repetitive nature of C. elegans postembryonic development is considered an oscillatory process, a concept that has gained traction from regulation by a circadian clock gene homologue. Nevertheless, each larval stage has a defined duration and entails specific events. Since the overall duration of development is controlled by numerous factors, we have asked whether different rate-limiting interventions impact all stages equally. RESULTS: We have measured the duration of each stage of development for over 2500 larvae, under varied environmental conditions known to alter overall developmental rate. We applied changes in temperature and in the quantity and quality of nutrition and analysed the effect of genetically reduced insulin signalling. Our results show that the distinct developmental stages respond differently to these perturbations. The changes in the duration of specific larval stages seem to depend on stage-specific events. Furthermore, our high-resolution measurement of the effect of temperature on the stage-specific duration of development has unveiled novel features of temperature dependence in C. elegans postembryonic development. CONCLUSIONS: Altogether, our results show that multiple factors fine tune developmental timing, impacting larval stages independently. Further understanding of the regulation of this process will allow modelling the mechanisms that control developmental timing.

A Unified Model for Entrainment by Circadian Clocks: Dynamic Circadian Integrated Response Characteristic (dCiRC).

Circadian rhythms are ubiquitous and are observed in all biological kingdoms. In nature, their primary characteristic or phenotype is the phase of entrainment. There are two main hypotheses related to how circadian clocks entrain, parametric and non-parametric models. The parametric model focuses on the gradual changes of the clock parameters in response to the changing ambient condition, whereas the non-parametric model focuses on the instantaneous change of the phase of the clock in response to the zeitgeber. There are ample empirical data supporting both models. However, only recently has a unifying model been proposed, the circadian integrated response characteristic (CiRC). In the current study, we developed a system of ordinary differential equations, dynamic CiRC (dCiRC), that describes parameters of circadian rhythms and predicts the phase of entrainment in zeitgeber cycles. dCiRC mathematically extracts the underlying information of velocity changes of the internal clock that reflects the parametric model and the phase shift trajectory that reflects the non-parametric model from phase data under entraining conditions. As a proof of concept, we measured clock parameters of 26 Neurospora crassa ecotypes in both cycling and constant conditions using dCiRC. Our data showed that the morning light shortens the period of the clock while the afternoon light lengthens it. We also found that individual ecotypes have different strategies of integrating light effects to accomplish the optimal phase of entrainment, a model feature that is consistent with our knowledge of how circadian clocks are organized and encoded. The unified model dCiRC will provide new insights into how circadian clocks function under different zeitgeber conditions. We suggest that this type of model may be useful in the advent of chronotherapies.

Weekly and seasonal variation in the circadian melatonin rhythm in humans: A response.

We read with interest the commentary by Skeldon and Dijk about our article "Weekly, seasonal and chronotype-dependent variation of dim light melatonin onset." The discussion points raised by Skeldon and Dijk are currently among the most hotly debated in human circadian science. What external factors determine human phase of entrainment? How great is the contribution of natural versus artificial light and sun time versus social time? Our intra-individual data add to the still limited evidence from field studies in this matter. In their commentary, Skeldon and Dijk formulate two either-or hypotheses, postulating that humans entrain either solely to the natural light-dark cycle (sun time referenced by midday) (H1 ) or solely to the light selected by local clock time and social constraints (H2 ). Neither hypothesis accounts for the effect of season on human light exposure. We interpreted our findings along more complex lines, speculating that the 1-h earlier melatonin rise in summer found in our sample is likely the combined result of daylight saving time (DST)-induced behavioral advances and a stronger natural zeitgeber in summer (light exposure determined by social and seasonal factors, Horiginal ). Here, we show how the criticism by Skeldon and Dijk is based on two sentences quoted out of context (misrepresenting our hypothesis as H1 ) and that their hypothesis H2  leaves out important seasonal components in light exposure.

Principles underlying the complex dynamics of temperature entrainment by a circadian clock.

Autonomously oscillating circadian clocks resonate with daily environmental (zeitgeber) rhythms to organize physiology around the solar day. Although entrainment properties and mechanisms have been studied widely and in great detail for light-dark cycles, entrainment to daily temperature rhythms remains poorly understood despite that they are potent zeitgebers. Here we investigate the entrainment of the chronobiological model organism Neurospora crassa, subject to thermocycles of different periods and fractions of warm versus cold phases, mimicking seasonal variations. Depending on the properties of these thermocycles, regularly entrained rhythms, period-doubling (frequency demultiplication) but also irregular aperiodic behavior occurs. We demonstrate that the complex nonlinear phenomena of experimentally observed entrainment dynamics can be understood by molecular mathematical modeling.

The Circadian Clock, the Brain, and COVID-19: The Cases of Olfaction and the Timing of Sleep.

Daily rhythms of behavior and neurophysiology are integral to the circadian clocks of all animals. Examples of circadian clock regulation in the human brain include daily rhythms in sleep-wake, cognitive function, olfactory sensitivity, and risk for ischemic stroke, all of which overlap with symptoms displayed by many COVID-19 patients. Motivated by the relatively unexplored, yet pervasive, overlap between circadian functions and COVID-19 neurological symptoms, this perspective piece uses daily variations in the sense of smell and the timing of sleep and wakefulness as illustrative examples. We propose that time-stamping clinical data and testing may expand and refine diagnosis and treatment of COVID-19.

A Timely Call to Arms: COVID-19, the Circadian Clock, and Critical Care.

We currently find ourselves in the midst of a global coronavirus disease 2019 (COVID-19) pandemic, caused by the highly infectious novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here, we discuss aspects of SARS-CoV-2 biology and pathology and how these might interact with the circadian clock of the host. We further focus on the severe manifestation of the illness, leading to hospitalization in an intensive care unit. The most common severe complications of COVID-19 relate to clock-regulated human physiology. We speculate on how the pandemic might be used to gain insights on the circadian clock but, more importantly, on how knowledge of the circadian clock might be used to mitigate the disease expression and the clinical course of COVID-19.

A circadian clock in a nonphotosynthetic prokaryote.

Circadian clocks create a 24-hour temporal structure, which allows organisms to occupy a niche formed by time rather than space. They are pervasive throughout nature, yet they remain unexpectedly unexplored and uncharacterized in nonphotosynthetic bacteria. Here, we identify in Bacillus subtilis circadian rhythms sharing the canonical properties of circadian clocks: free-running period, entrainment, and temperature compensation. We show that gene expression in B. subtilis can be synchronized in 24-hour light or temperature cycles and exhibit phase-specific characteristics of entrainment. Upon release to constant dark and temperature conditions, bacterial biofilm populations have temperature-compensated free-running oscillations with a period close to 24 hours. Our work opens the field of circadian clocks in the free-living, nonphotosynthetic prokaryotes, bringing considerable potential for impact upon biomedicine, ecology, and industrial processes.

Weekly, seasonal, and chronotype-dependent variation of dim-light melatonin onset.

In humans, the most important zeitgeber for entrainment is light. Laboratory studies have shown that meaningful changes in light exposure lead to phase shifts in markers of the circadian clock. In natural settings, light is a complex signal varying with external conditions and individual behaviors; nonetheless, phase of entrainment is assumed to be fairly stable. Here, we investigated the influence of season and weekly schedule (as indicators of variation in light landscapes) on phase of entrainment. Using a within-subjects design (N = 33), we assessed dim-light melatonin onset (DLMO) as a circadian phase marker in humans, on workdays and work-free days, in summer (under daylight saving time) and in winter, while also estimating sleep times from actimetry. Our mixed-model regressions show that both season and weekly structure are linked with changes in phase of entrainment and sleep. In summer, both DLMO and sleep times were about 1 hour earlier compared to winter, and sleep duration was shorter. On work-free days, DLMO and sleep times were later, and their phase relationship differed more relative to workdays. All these effects were stronger in later chronotypes (those who habitually sleep late). Our results confirm that phase of entrainment is earlier when stronger zeitgebers are present (summer) and show that it relates to midday or midnight rather than sunrise or sunset. Additionally, they suggest that late chronotypes are capable of rapid phase shifts each week as they move between workdays and work-free days, stimulating interesting questions about the stability of circadian phase under natural conditions.

Circadian effects on stroke outcome - Did we not wake up in time for neuroprotection?

The occurrence of stroke in humans peaks in the morning. A recent study revealed that time of day mitigates the therapeutic impact of neuroprotective paradigms. These findings might not only explain the previous failure of translation of neuroprotective therapies but inspire new paradigms in stroke chronopathophysiology research. Taking chronotype into account may complement the many factors that influence efficacy of experimental therapies in stroke.

A functional context for heterogeneity of the circadian clock in cells.

Characterization of circadian systems at the organism level-a top-down approach-has led to definition of unifying properties, a hallmark of the science of chronobiology. The next challenge is to use a bottom-up approach to show how the molecular workings of the cellular circadian clock work as building blocks of those properties. We review new studies, including a recently published PLOS Biology paper by Nikhil and colleagues, that show how programmed but also stochastic generation of variation in cellular circadian period explain important adaptive features of entrained circadian phase.

Prolonged quiescence delays somatic stem cell-like divisions in Caenorhabditis elegans and is controlled by insulin signaling.

Cells can enter quiescence in adverse conditions and resume proliferation when the environment becomes favorable. Prolonged quiescence comes with a cost, reducing the subsequent speed and potential to return to proliferation. Here, we show that a similar process happens during Caenorhabditis elegans development, providing an in vivo model to study proliferative capacity after quiescence. Hatching under starvation provokes the arrest of blast cell divisions that normally take place during the first larval stage (L1). We have used a novel method to precisely quantify each stage of postembryonic development to analyze the consequences of prolonged L1 quiescence. We report that prolonged L1 quiescence delays the reactivation of blast cell divisions in C. elegans, leading to a delay in the initiation of postembryonic development. The transcription factor DAF-16/FOXO is necessary for rapid recovery after extended arrest, and this effect is independent from its role as a suppressor of cell proliferation. Instead, the activation of DAF-16 by decreased insulin signaling reduces the rate of L1 aging, increasing proliferative potential. We also show that yolk provisioning affects the proliferative potential after L1 arrest modulating the rate of L1 aging, providing a possible mechanistic link between insulin signaling and the maintenance of proliferative potential. Furthermore, variable yolk provisioning in embryos is one of the sources of interindividual variability in recovery after quiescence of genetically identical animals. Our results support the relevance of L1 arrest as an in vivo model to study stem cell-like aging and the mechanisms for maintenance of proliferation potential after quiescence.

Strategies to decrease social jetlag: Reducing evening blue light advances sleep and melatonin.

The timing of sleep is under the control of the circadian clock, which uses light to entrain to the external light-dark cycle. A combination of genetic, physiological and environmental factors produces individual differences in chronotype (entrained phase as manifest in sleep timing). A mismatch between circadian and societal (e.g., work) clocks leads to a condition called social jetlag, which is characterized by changing sleep times over work and free days and accumulation of sleep debt. Social jetlag, which is prevalent in late chronotypes, has been related to several health issues. One way to reduce social jetlag would be to advance the circadian clock via modifications of the light environment. We thus performed two intervention field studies to describe methods for decreasing social jetlag. One study decreased evening light exposure (via blue-light-blocking glasses) and the other used increased morning light (via the use of curtains). We measured behaviour as well as melatonin; the latter in order to validate that behaviour was consistent with this neuroendocrinological phase marker of the circadian clock. We found that a decrease in evening blue light exposure led to an advance in melatonin and sleep onset on workdays. Increased morning light exposure advanced neither melatonin secretion nor sleep timing. Neither protocol led to a significant change in social jetlag. Despite this, our findings show that controlling light exposure at home can be effective in advancing melatonin secretion and sleep, thereby helping late chronotypes to better cope with early social schedules.

Tardiness Increases in Winter: Evidence for Annual Rhythms in Humans.

Annual rhythms in humans have been described for a limited number of behavioral and physiological parameters. The aim of this study was to investigate time-of-year variations in late arrivals, sick leaves, dismissals from class (attendance), and grades (performance). Data were collected in Dutch high school students across 4 academic years (indicators of attendance in about 1700 students; grades in about 200 students). Absenteeism showed a seasonal variation, with a peak in winter, which was more strongly associated with photoperiod (number of hours of daylight) compared with other factors assessed (e.g., weather conditions). Grades also varied with time of year, albeit differently across the 4 years. The observed time-of-year variation in the number of sick leaves was in accordance with the literature on the seasonality of infectious diseases (e.g., influenza usually breaks out in winter). The winter peak in late arrivals was unexpected and requires more research. Our findings could be relevant for a seasonal adaptation of school schedules and working environments (e.g., later school and work hours in winter, especially at higher latitudes where seasonal differences in photoperiod are more pronounced).

Are There Circadian Clocks in Non-Photosynthetic Bacteria?

Circadian clocks in plants, animals, fungi, and in photosynthetic bacteria have been well-described. Observations of circadian rhythms in non-photosynthetic Eubacteria have been sporadic, and the molecular basis for these potential rhythms remains unclear. Here, we present the published experimental and bioinformatical evidence for circadian rhythms in these non-photosynthetic Eubacteria. From this, we suggest that the timekeeping functions of these organisms will be best observed and studied in their appropriate complex environments. Given the rich temporal changes that exist in these environments, it is proposed that microorganisms both adapt to and contribute to these daily dynamics through the process of temporal mutualism. Understanding the timekeeping and temporal interactions within these systems will enable a deeper understanding of circadian clocks and temporal programs and provide valuable insights for medicine and agriculture.